w
—
'
140
FREEZE-DRYÏNG
J. Controed Dehydmüon Prior to Freezing
.
Water content was
di‘ereny in pancreas and in marine €ggs. In pancreas, water
…
_
was removed by placihg thetisSue-for periods of 15, ’30, 45 and—60 seconds in full strength
glycerol prior to-freezing. A‘ drop of glycerol was placed on a formvar loop and a 0_1, mm
to 0.2 mm piece 'of pancreas placed in it. The ldop was mounted in the injector and at the
,.
required time, the assembly-Shot into the quenching uid; This method gives some control
over watérwèontent since ‘gly‘c‘etol is not eXpected to penetrate into the tissue in the short :
periods used-before freezing, the, primary result being tissue shrikage as demonstrated
by Luyet and Keane:(l953). However, “the tissue Was by nomeans uniform and homogenous
_
in
water deprivati0ni : !
.
,
'
,
'
-
'
'
'
-
'
,
:
…
Marine. eggsact essentially as osmometèrs oVer a certain range of external salt concen—
trations (Churney, 1940) and so may be osmotically dehydrated With much better control
”
of Water c0ntent than ‘can‘ be obtained 'ipancreas by the»tech‘niqueS’-described above.
…
Unfertizèd “ and
eggS of Spisula Were used.- Egg diameter—s
were checked and it was found that egg volume was approximately proportional
_
cdnc‘entration down to approximately double strength sea“ Water. At higher concentrations,
théeggs stillshrank but.‘thevitelline membrane showed much cnkling and would usually
_
various î‘points.ïlnearlÿ experiments we went no lower than
100430 seawater (ICO—x means ”normal Sea Wat‘erfr0m which-xf‘% of the water has been
removed) but soon found that in no case were able to avoid ice crystal formation. Figure 2
—
isa micrograph taken from a, Cell equilibrated in 100—30‘ sea water. The endoplasmic reti,
colum, mitochondria, vitelline membrane, etc., are clearly identiable. However, the cyto,:Ï
plasm is Clearly similar to that of Figure 1, that is, possesses ice crystals (or the remains of
—
such after dehydration). In unfertilized eggs it was necessary to go to at least 100-50 and
‘
usually ICO-60 (that is, greater thandouble strength sea water) in order to avoid ice crystals.
of dehydration necessary was ‘eVen greater :
that is,
cells were—“amy ConsiSte11tly f0und in _3x sea Water,“ that is, iGO-67,
tous a most surprising 'reSult. Even more surprisingwas
degree cf preservation of
“dehydration. As can be seen in Figures 54, 5 and 6 of partliènogenetrcallyacüvated eggs, ’thäàgmitobhbndrîe‘r look surprisinglynormal eXcept that the
matrix
With most Stains
crista‘e appear as if the
space between its two Components has disappeare‘d, (Figure ‘4)‘thàtiS,à‘S‘ if two unit mem-‘
,
…
opposed along their length. We Will see this later in rat liver and pancreas.
ribŒomes can easily be Seen in many plaCes and are usually in the neighb0rhood of 2501
$
3,00, À that—is, ,largerthan
xed material,reticulum,
to bé” composed ofumf membranes
The most SpcÇ{
‘
tacular structures are the microtubules Which lie
in bundles in the cytoplasm and the
ciàted ribosonies.The latter are very regularly packed in almost all regions possessing
…
,
microtubules and in some regions this regular, hexagonal packing may involve
”
‘
«section.: The,
fêä9ië
ones; thèther
.
,
thlsememe
,1Howevernceus
.
whlchhavenot
ice crystals but,,m ïwhieh;ÿ
seen:_iurzassociation With;
‘
environment of the micfotubule
such as to
—
'
140
FREEZE-DRYÏNG
J. Controed Dehydmüon Prior to Freezing
.
Water content was
di‘ereny in pancreas and in marine €ggs. In pancreas, water
…
_
was removed by placihg thetisSue-for periods of 15, ’30, 45 and—60 seconds in full strength
glycerol prior to-freezing. A‘ drop of glycerol was placed on a formvar loop and a 0_1, mm
to 0.2 mm piece 'of pancreas placed in it. The ldop was mounted in the injector and at the
,.
required time, the assembly-Shot into the quenching uid; This method gives some control
over watérwèontent since ‘gly‘c‘etol is not eXpected to penetrate into the tissue in the short :
periods used-before freezing, the, primary result being tissue shrikage as demonstrated
by Luyet and Keane:(l953). However, “the tissue Was by nomeans uniform and homogenous
_
in
water deprivati0ni : !
.
,
'
,
'
-
'
'
'
-
'
,
:
…
Marine. eggsact essentially as osmometèrs oVer a certain range of external salt concen—
trations (Churney, 1940) and so may be osmotically dehydrated With much better control
”
of Water c0ntent than ‘can‘ be obtained 'ipancreas by the»tech‘niqueS’-described above.
…
Unfertizèd “ and
eggS of Spisula Were used.- Egg diameter—s
were checked and it was found that egg volume was approximately proportional
_
cdnc‘entration down to approximately double strength sea“ Water. At higher concentrations,
théeggs stillshrank but.‘thevitelline membrane showed much cnkling and would usually
_
various î‘points.ïlnearlÿ experiments we went no lower than
100430 seawater (ICO—x means ”normal Sea Wat‘erfr0m which-xf‘% of the water has been
removed) but soon found that in no case were able to avoid ice crystal formation. Figure 2
—
isa micrograph taken from a, Cell equilibrated in 100—30‘ sea water. The endoplasmic reti,
colum, mitochondria, vitelline membrane, etc., are clearly identiable. However, the cyto,:Ï
plasm is Clearly similar to that of Figure 1, that is, possesses ice crystals (or the remains of
—
such after dehydration). In unfertilized eggs it was necessary to go to at least 100-50 and
‘
usually ICO-60 (that is, greater thandouble strength sea water) in order to avoid ice crystals.
of dehydration necessary was ‘eVen greater :
that is,
cells were—“amy ConsiSte11tly f0und in _3x sea Water,“ that is, iGO-67,
tous a most surprising 'reSult. Even more surprisingwas
degree cf preservation of
“dehydration. As can be seen in Figures 54, 5 and 6 of partliènogenetrcallyacüvated eggs, ’thäàgmitobhbndrîe‘r look surprisinglynormal eXcept that the
matrix
With most Stains
crista‘e appear as if the
space between its two Components has disappeare‘d, (Figure ‘4)‘thàtiS,à‘S‘ if two unit mem-‘
,
…
opposed along their length. We Will see this later in rat liver and pancreas.
ribŒomes can easily be Seen in many plaCes and are usually in the neighb0rhood of 2501
$
3,00, À that—is, ,largerthan
xed material,reticulum,
to bé” composed ofumf membranes
The most SpcÇ{
‘
tacular structures are the microtubules Which lie
in bundles in the cytoplasm and the
ciàted ribosonies.The latter are very regularly packed in almost all regions possessing
…
,
microtubules and in some regions this regular, hexagonal packing may involve
”
‘
«section.: The,
fêä9ië
ones; thèther
.
,
thlsememe
,1Howevernceus
.
whlchhavenot
ice crystals but,,m ïwhieh;ÿ
seen:_iurzassociation With;
‘
environment of the micfotubule
such as to